Process for producing nerve stem cells, motor neurons, and GABAergic neurons from embryonic stem cells

ABSTRACT

The present invention provides a method for producing motor neurons and GABAergic neurons characterized by including suspension-culturing embryonic stem cells in the presence or absence of a protein noggin to form embryoid bodies, selectively amplifying into neural stem cells from them by suspension culture in the presence of a fibroblast growth factor and a sonic hedgehog protein, and then differentiating the same. According to this method, at least motor neurons and GABAergic neurons can be systemically and efficiently produced from ES cells. Selective acquisition of neurons would be applicable to transplant therapy for amyotrophic lateral sclerosis, Huntington&#39;s chorea, Alzheimer&#39;s disease, etc.

TECHNICAL FIELD

The present invention relates to a method for selectively producingneural stem cells from embryonic stem cells (ES cells) and also to amethod for selectively and efficiently producing motor neurons andGABAergic neurons.

BACKGROUND OF THE INVENTION

In the central nervous system of a mammal, neural stem cells existthrough the entire life of the individual and contribute to the growthand homeostasis of the central nervous system by producing a variety ofneurons and glia. Techniques that have recently been developed aiming atthe isolation and culturing of neural stem cells from the brain ofmammals, including humans, are expected to provide potentialapplications to the cell transplant therapies for various types ofneurodegenerative diseases and injures. No appreciable achievement isstill reported, however, despite some attempts exerted to obtain, fromthe neural stem cells cultured and amplified in vitro, different typesof neurons that can be generated and differentiated from stem cellsunder control of diversified endogenous and exogenous factors,espeGially motor neurons that can be specifically generated at theinitial stage of embryogenesis.

Accordingly, an object of the present invention is to provide means forefficiently inducing differentiation of ES cells, which have thecapacity to differentiate into any type of mature cells in anindividual, into neural stem cells maintaining properties of those cellsin the early stage of development. Another object of the presentinvention is to provide a technique for selectively producing a specifictype of neuron, such as motor neurons, from the neural stem cells.

DISCLOSURE OF THE INVENTION

The present inventors have investigated a variety of conditions underwhich it is necessary for embryoid bodies to be generated from ES cells,and for ES cells to differentiate and be induced into neural stem cellsand eventually become the neurons. As a result, it has been found thatthe presence of noggin protein has a particularly important role in theinduction of neural stem cells within embryoid bodies derived from EScells; the use of a medium containing a fibloblast growth factor (FGF)and a sonic hedgehog protein is extremely efficient for amplifyingneutral stem cells emerging in embryoid bodies; and when such neutralstem cells are differentiated, motor neurons and GABAergic neurons canbe produced selectively and efficiently. Thus, the present invention hasbeen accomplished on the basis of these findings.

Accordingly, the present invention provides a method for formingembryoid bodies, characterized by subjecting ES cells to suspensionculture in the presence of noggin protein.

The present invention also provides a method for producing neural stemcells, characterized by subjecting ES cells to suspension culture in thepresence or absence of noggin protein, to thereby form embryoid bodies,and subsequently subjecting the embryoid bodies to suspension culture inthe presence of fibroblast growth factor and sonic hedgehog protein.

The present invention also provides a method for producing motor neuronsand GABAergic neurons, characterized by subjecting ES cells tosuspension culture in the presence or absence of noggin protein, tothereby form embryoid bodies, and subsequently subjecting the embryoidbodies to suspension culture in the presence of fibroblast growth factorand sonic hedgehog protein, to thereby induce neural stem cells, anddifferentiate the resultant neural stem cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relation between days of culturing embryoid bodies andformation of neurospheres.

FIG. 2 shows an image of immunostaining of neurospheres afterdifferentiation. The stained region shows expression of β-III-tubulin,which indicates a neuron.

FIG. 3 shows another image of immunostaining of differentiatedneurospheres by anti-1st-1 and anti-ChAT (choline acetyltransferase)antibodies which are markers for motor neurons.

FIG. 4 shows yet another image of immunostaining of differentiatedneurospheres by anti-GAD (glutamic acid decarboxylase) 67 antibody.

FIG. 5 shows an image of immunostaining of differentiated neurospheresthat have undergone subculture. Cells were immunolabeled by anti-β-III,GFAP, and 04 antibodies.

FIG. 6 shows the percentage of neurons and glia cells after subcultureof neurospheres.

FIG. 7 shows the effect of addition of a noggin protein.

FIG. 8 shows the effect of addition of a sonic hedgehog protein.

BEST MODE FOR CARRYING OUT THE INVENTION

The ES cells used in the present invention may be those which-havealready been established as cultured cells. For example, ES cell linesfrom mice, hamsters, pigs, and humans may be employed. Specific examplesinclude 129/O1a-mouse-derived ES cells, such as EB3 and E14tg2.Preferably, the ES cells are subcultured in a GMEM medium or a similarmedium supplemented with serum.

In the formation of embryoid bodies from ES cells, suspension culture ofES cells in a medium to which noggin protein has been added is effectivefor promoting differentiation-inducing efficiency from ES cells toneural stem cells. The noggin protein may be a Xenopus noggin protein.Alternatively, full-length cDNA of Xenopus noggin is transferred to COS7cells, followed by culturing to cause transient expression of the nogginprotein, and the resultant supernatant may be used as is. Preferably,the concentration of the noggin protein in medium is 1 to 50% (v/v) orthereabouts in terms of the volume of culture supernatant. Suspensionculture of ES cells is performed by use of serum-containing α-MEM mediumfor 4 to 8 days at a concentration of approximately 1×10⁵ ES cells/mL.Examples of useful sera include bovine serum and pig serum. The serumconcentration is 5 to 15%, preferably 8 to 12%. Preferably,2-mercaptoethanol is added to the α-MEM medium in such an amount thatachieves a concentration of 0.01 to 0.5 mM, particularly 0.05 to 0.2 mM.The culturing is preferably performed in 5% CO₂, at 35-40° C.

It is highly preferred that the noggin protein be added during formationof embryoid bodies; i.e., during the period from day 0 to day 6 ofculturing.

In order to amplify neural stem cells which have been obtained from EScells via the above-prepared embryoid bodies, suspension culture isperformed by use of a neural stem cell amplification medium containingnot only a fibroblast growth factor but also a sonic hedgehog protein.The addition of sonic hedgehog protein promotes efficiency of inducingdifferentiation of neural stem cells to motor neuron precursors, andalso improves multiplication efficiency of the neural stem cells.Moreover, through subsequent differentiation culturing, the neural stemcells are in fact differentiated into motor neurons and GABAergicneurons.

A preferred fibroblast growth factor (FGF) is FGF-2. The FGF content ofthe medium is preferably 5 to 50 ng/mL, more preferably 10 to 40 ng/mL.Examples of preferred sonic hedgehog proteins include mouse sonichedgehog protein. The sonic hedgehog protein content of the medium is 1to 20 nM, preferably 1 to 10 nM.

The medium is preferably a DMEM medium containing, in addition to theaforementioned components, glucose, glutamine, insulin, transferrin,progesterone, putrecine, selenium chloride, heparin, etc. Use of aDMEM:F12 medium is particularly preferred. The culturing is preferablyperformed in 5% CO₂, at 35-40° C., for a period of 7 to 9 days.

Through the above-described suspension culture, single-cell-derived,aggregated masses of cells, called neurospheres, are formed.

The thus-obtained neurospheres have originated solely from neural stemcells, and thus the above-mentioned culture method is proven to attainvery high differentiation efficiency.

When the thus-obtained neural stem cells are cultured in an ordinarydifferentiation medium, differentiation into only motor neurons andGABAergic neurons alone is induced. Here, a preferreddifferentiation-inducting medium is a DMEM:F12 medium containingglucose, glutamine, insulin, transferrin, progesterone, putrecine, andselenium chloride (in other words, a medium designed for amplifyingneural stem cells but excluding FGF and heparin). In this medium, sonichedgehog protein may or may not be present. The culturing is preferablyperformed in 5% CO₂, at 35-40° C., for 5 to 7 days.

Neural cells obtained from ES cells through conventional techniques ofdifferentiation contain not only neurons but also significant amounts ofglia cells, among other cells. Thus, heretofore, they have only limitedusage value. In contrast, wherein the neurons obtained by working thepresent invention are substantially formed only of motor neurons andGABAergic neurons.

EXAMPLES

The present invention will next be described by way of examples, whichshould not be construed as limiting the invention thereto.

A. Materials and methods

(1) Culture-Passage of Mouse ES Cells and Formation of Embryoid Bodies

E14tg2a ES cells derived from 129/O1a mice and EB3 ES cells (which allowselection of undifferentiated ES cells through insertion ofblasticidin-resistant gene to the Oct3/4 locus of E14tg2a) weresubcultured by a routine method in a GMEM medium (Glasgow minimumessential medium) containing 10% fetal calf serum, nonessential aminoacids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, and 1,000 U/mLleukemia inhibitory factor (LIF). The culture conditions were 5% CO₂ at37° C. (hereafter, when “culture” is referred to, these conditionsapply).

Formation of embryoid bodies (EBs) from the ES cells was carried out asfollows. Firstly, ES cells were washed with PBS. Subsequently, thewashed cells were treated with 0.25% trypsin—1 mM EDTA, and then thetreatment reaction was stopped. The cells were dissociated by pipetting,and seeded in a bacterial culture dish filled with α-MEM mediumcontaining 10% fetal calf serum and 0.1 mM 2-mercaptoethanol. In thepresence or absence of noggin protein, suspension culture was performedfor 4 to 8 days, whereby EBs were formed. The noggin protein employedwas a culture supernatant of COS7 cells to which full-length cDNA ofXenopus noggin had been introduced for transitory expression.

(2) Isolation of Neural Stem Cells by Selective Culture of EBs

The EBs formed as described above, together with the culture liquid,were transferred to a centrifuge tube. The tube was allowed to stand for10 minutes, so that the EBs were sedimented at the bottom. Thesupernatant was removed, and the EBs were re-suspended in PBS. The testtube was allowed to stand for 10 minutes again. The supernatant wasremoved, and the EBs were re-suspended in a solution containing 0.25%trypsin and 1 mM EDTA PBS, followed by incubation at 37° C. for fiveminutes. The protein degradation reaction was stopped by use of α-MEMmedium containing 10% fetal calf serum. The cells were dissociated bypipetting. The dissociated cells were centrifugally washed with α-MEMmedium twice, and seeded at a concentration of 5×10⁴ cells/mL in eitherof the following mediums designed for neural stem cell amplification: a1:1 medium of DMEM (Dulbecco's modified Eagle's medium) and F12, wherethe DMEM had been supplemented with glucose (0.6%), glutamine (2 mM),insulin (25 μg/mL), transferrin (100 μg/mL), progesterone (20 nM),putrecine (60 μM), selenium chloride (30 nM), FGF-2 (20 ng/mL), andheparin (2 μg/mL); or the same medium but further containing a mousesonic hedgehog (5 nM), followed by suspension culture for 7 to 9 days,whereby neurospheres (cell clusters derived from a single cell) wereformed. The neurospheres were centrifugally washed with adifferentiation medium containing neither FGF-2 nor heparin, and thewashed cells—in the “as washed” state or after dissociated throughpipetting—were seeded in a culture petri dish coated withpoly-L-ornithine and filled with a differentiation medium, wherebydifferentiation is allowed to proceed in the presence or absence of asonic hedgehog protein (5 nM) for 5 to 7 days. Separately, theabove-obtained neurospheres were again dissociated into single cells,subcultured in a medium designed for amplification of neural stem cells,to thereby form secondary neurospheres. The thus-obtained secondaryneurospheres are also caused to differentiate as described above.

(3) Identification of Differentiated Neurons and Glia Cells ThroughImmunostaining

The thus-differentiated neurons and glia cells were identified by aroutine immunostaining method using a fluorescent antibody. Motorneurons were identified by mouse anti-Isl-1 monoclonal antibody, goatanti-ChAT polyclonal antibody, and mouse anti-β-III tublin monoclonalantibody; and GABAergic neurons were identified by rabbit anti-GAD67polyclonal antibody. Regarding glia cells, astrocytes were identified byrabbit anti-GFAP polyclonal antibody, and oligodendrocytes wereidentified by mouse anti-04 monoclonal antibody.

B. Test results

(1) Isolation and Purification of Neural Stem Cells by Selective Cultureof EBs

Firstly, the inventors focused on the initial stage of differentiationof ES cells via formation of EBs, and investigated as to when neuralstem cells emerged during culture. Specifically, EBs which had undergone4 to 8 days of culture were dissociated into single cells, followed byculture for 7 days in a medium designed for amplifying neural stemcells, whereby neurospheres were formed. The neurospheres weretransferred to a differentiation medium, and allowed to differentiate.Thereafter, their differentiation capacity was checked. Also,neurospheres were subcultured for checking their self-renewal capacity.

FIG. 1 shows the results of selective culture of neural stem cells (theneurosphere method), wherein 6 or 8 days after start of EB formationthrough suspension culture, the formed EBs were dissociated into singlecells and subjected to the neurosphere method. The number of the neuralstem cells emerged in the EBs was taken as that of the obtainedneurospheres. Neural stem cells (capable of forming neurospheres) whichwere to be identified by the present method were virtually not detecteduntil day 4 of culture. On day 6 of culture, neural stem cells accounted0.25% of all the cells, and on day 8, neural stem cells accounted 1.1%,thus gradual increase in cell count was acknowledged.

The neurospheres obtained from the EBs on day 6 (see FIG. 1) werecultured for 7 days under differentiation conditions, and theirdifferentiation capacity was checked through immunostaining. The resultsare shown in FIGS. 2 to 4. When triple immunostaining was performed byuse of β-III-tubulin (a marker for neurons) and GFAP and anti-04antibody (markers for glial cells), virtually all neurospheres werefound to be formed only of neurons, which express β-III-tubulin, and noglial cells were detected (FIG. 2). The neurons were found to contain atleast motor neurons expressing at least Isl-1 and ChAT (note: the motorneurons are seen in FIG. 3 as round images and fibrous images) andGABAergic neurons expressing GAD67 (note: the GABAergic neurons are seenin FIG. 4 as fibrous images).

Moreover, the obtained neurospheres were subjected to subculture, tothereby obtain secondary neurospheres. The secondary neurospheres werecultured for 7 days under differentiation conditions, and theirdifferentiation capacity was checked through immunostaining. As aresult, all the neurospheres were found to contain glia cells (FIG. 5);with 84.2% thereof containing both neurons and glias (FIG. 6). FIG. 5shows the results of triple immunostaining with β-III-tubulin (definitethin fibers), GFAP (portions surrounding those of β-III-tubulin), andanti-04 antibody (portions surrounding those of GFAP).

As a result, the following was confirmed: When neurospheres aredissociated into single cells and then subcultured to thereby causeformation of new neurospheres and differentiation, most clones thereofcontain both neurons and glias, and like the case in which glia cellsemerge in a later period in development of actual central nervoussystem, neural stem cells isolated from EBs, after undergoingsubculture, also exhibit pluripotent capacity.

(2) Improvement of Efficiency in Inducing Neural Stem CellDifferentiation by Use of Noggin Protein

In an attempt to improve efficiency in inducing neural stem celldifferentiation, during EB formation (6 days), noggin protein was added.The noggin protein employed was in the form of solution prepared by useof the supernatant of the culture in which full-length cDNA of Xenopuswas inserted into a pEF-BOS expression vector and then transfected intoCOS7 cells for transient expression. The control employed was asupernatant of culture of COS7 cells to which only the expression vectorhad been incorporated. As shown in FIG. 7, the number of neurospheresformed of neural stem cells and induced to differentiate among EBsincreases with the volume of the noggin culture supernatant, reaching apeak at 1/10 in volume.

(3) Improvement in Efficiency of Motor Neuron Differentiation by Use ofSonic Hedgehog Protein

In an attempt to improve efficiency of motor neuron production anddifferentiation from EB-derived neural stem cells, sonic hedgehogprotein was added to proliferating neural stem cells, in other words,during formation of primary culture neurospheres derived from EBs, andthe effect of the addition was studied. After the neurospheres weredissociated into single cells and cultured for 5 days in adifferentiation medium, motor neurons were identified through doubleimmunostaining by use of Isl-1 and β-III-tublin, and the number thereofwas quantified. As shown in FIG. 8, production of motor neurons doubledas a result of addition of 5 nM sonic hedgehog protein. When sonichedgehog protein was added to the differentiation medium in which neuraldifferentiation took place, no effect of addition was observed.

INDUSTRIAL APPLICABILITY

The present invention has thus found that ES cells have capability ofproducing at least motor neurons and GABAergic neurons systematicallyand efficiently. It also suggests that if neurons are selectivelyobtained therefrom, ES cells might make it possible to bring thepotential use to transplant therapies for amyotrophic lateral sclerosis,Huntinton's chorea, Alzheimer's disease, etc.

1. A method for forming embryoid bodies, which comprises subjecting embryonic stem cells to suspension culture in the presence of a noggin protein, wherein said noggin protein is added to the culture prior to formation of embryoid bodies.
 2. A method for producing neural stem cells, which comprises subjecting embryonic stem cells to suspension culture in the presence of a noggin protein, to thereby form embryoid bodies, and subsequently subjecting the embryoid bodies to suspension culture in the presence of a fibroblast growth factor and a sonic hedgehog protein to thereby form a culture of neural stem cells.
 3. The method according to claim 2, wherein concentration of the fibroblast growth factor in culture medium is 5 to 50 ng/mL, and that of the sonic hedgehog protein in culture medium is 1 to 20 nM.
 4. A method for producing motor neurons and GABAergic neurons, which comprises subjecting ES cells to suspension culture in the presence of a noggin protein, to thereby form embryoid bodies, and subsequently subjecting the embryoid bodies to suspension culture in the presence of a fibroblast growth factor and a sonic hedgehog protein, to thereby induce formation of neural stem cells, and differentiating the resultant neural stem cells in a differentiation medium.
 5. The method according to claim 4, wherein concentration of the fibroblast growth factor in culture medium is 5 to 50 ng/mL, and that of the sonic hedgehog protein in culture medium is 1 to 20 nM.
 6. The method according to claim 4, wherein the resultant neurons are substantially formed of motor neurons and GABAergic neurons.
 7. The method according to claim 5, wherein the resultant neurons are substantially formed of motor neurons and GABAergic neurons.
 8. The method according to claim 2, wherein the culture of neural stem cells comprises neurospheres. 